Supramolecular Chiral Binding Affinity‐Achieved Efficient Synergistic Cancer Therapy

Abstract Supramolecular chirality‐mediated selective interaction among native assemblies is essential for precise disease diagnosis and treatment. Herein, to fully understand the supramolecular chiral binding affinity‐achieved therapeutic efficiency, supramolecular chiral nanoparticles (WP5⊃D/L‐Arg+DOX+ICG) with the chirality transfer from chiral arginine (D/L‐Arg) to water‐soluble pillar[5]arene (WP5) are developed through non‐covalent interactions, in which an anticancer drug (DOX, doxorubicin hydrochloride) and a photothermal agent (ICG, indocyanine green) are successfully loaded. Interestingly, the WP5⊃D‐Arg nanoparticles show 107 folds stronger binding capability toward phospholipid‐composed liposomes compared with WP5⊃L‐Arg. The enantioselective interaction further triggers the supramolecular chirality‐specific drug accumulation in cancer cells. As a consequence, WP5⊃D‐Arg+DOX+ICG exhibits extremely enhanced chemo‐photothermal synergistic therapeutic efficacy (tumor inhibition rate of 99.4%) than that of WP5⊃L‐Arg+DOX+ICG (tumor inhibition rate of 56.4%) under the same condition. This work reveals the breakthrough that supramolecular chiral assemblies can induce surprisingly large difference in cancer therapy, providing strong support for the significance of supramolecular chirality in bio‐application.

that, the pH of each solution was adjusted to 7.4.The as prepared WP5⊃D/DL/L-Arg solutions were stored at room temperature for future use.

Preparation of liposome.
Liposomes were prepared by thin film dispersion.Specifically, soy lecithin and cholesterol (5:1 mass ratio) were dissolved in chloroform.
The solvent is then removed with a rotary evaporator.Subsequently, 10 mL of ultrapure water was added, and the solution was stirred at 52 ℃ for 3 hours.After that, the solution was ultrasonicated for 10 min, resulting the liposome dispersion.
Preparation of WP5⊃D/DL/L-Arg+DOX.To prepare WP5⊃D/DL/L-Arg+DOX, DOX (molar ratio of DOX:WP5⊃D/DL/L-Arg = 1:2) was added to 10 mL of WP5⊃D/DL/L-Arg solution, respectively, and stirred for 24 hours.After that, WP5⊃D/DL/L-Arg+DOX solution was loaded into dialysis bag (molecular weight cutoff 14000), and was dialyzed in ultrapure water to remove the unloaded DOX.The concentration of WP5⊃D/DL/L-Arg+DOX mentioned in this work is referred to the concentration of Arg.
Preparation of WP5⊃D/DL/L-Arg+ICG.To prepare WP5⊃D/DL/L-Arg+ICG, ICG (molar ratio of ICG:WP5⊃D/DL/L-Arg = 1:2) was added to 10 mL of WP5⊃D/DL/L-Arg solution, respectively, and stirred for 24 hours.After that, WP5⊃D/DL/L-Arg+ICG solution was loaded into dialysis bag (molecular weight cutoff 14000), and DOX releasing behavior.To study the release behavior of DOX, 3 mL of WP5⊃D/L-Arg+DOX+ICG solution was placed into a dialysis bag, and then the dialysis bag was placed into a 50 mL centrifuge tube, which was pre-filled with 30 mL PBS buffer solution (pH 7.4 or 6.5).The centrifuge tube was kept shaking by a constant temperature shaker at 37 ℃ and 118 rpm.At various time periods, the UV-Vis absorption of DOX at 478 nm and ICG at 780 nm of the solution outside the dialysis bag were determined by UV spectrophotometer.After the measurement, the solution was poured back into the centrifuge tube.To study the drug release behavior under NIR light irradiation, the above experiments were repeated under 808 nm laser irradiation.The releasing efficiency of DOX was calculated according to the standard calibration curve of DOX in PBS.(pH = 7.4: yDOX = 0.01642x − 0.00247; pH = 6.5: yDOX = 0.01640x − 0.02188,) Cellular uptake behavior.To investigate the effect of supramolecular chirality of WP5⊃D/DL/L-Arg+DOX+ICG on cellular uptake, CT26 cells were inoculated onto a confocal culture dish for 12 hours.Subsequently, WP5⊃D/DL/L-Arg+DOX+ICG were added.After 24 hours, the cells were washed with PBS for three times and the nuclei were stained with DAPI.Eventually, confocal fluorescence microscopy was used to observe the uptake behavior of WP5⊃D/DL/L-Arg+DOX+ICG.
To explore the cellular uptake mechanism of WP5⊃D/DL/L-Arg+DOX+ICG into CT26 cells, CT26 cells were seeded on a confocal dish for 12 hours, and then placed in a refrigerator at 4 ℃ for 1 hour, Then WP5⊃D/DL/L-Arg+DOX+ICG were added and continued to incubate in a refrigerator at 4 ℃ for 24 hours.The control group was also added with the same concentration of WP5⊃D/DL/L-Arg+DOX+ICG, and incubated at 37 ℃ for 24 hours.After incubation, the cells were washed three times with PBS and the nuclei were stained with DAPI for 20 minutes.The cellular uptake results were then observed by confocal fluorescence microscopy.
To quantify the mean fluorescence intensity of WP5⊃D/DL/L-Arg+DOX+ICG treated CT26 cells by flow cytometry, the CT26 cells were inoculated in a 6-well plate at a density of 1 × 10 5 cells per well at 37 °C and 5% CO2 for 24 hours.Then, the CT26 cells were incubated with WP5⊃D/DL/L-Arg+DOX+ICG (40 µg mL −1 , 37 °C, 24 hours), and the cell culture medium was sucked up 24 hours later followed with PBS washing.The cells with various treatments were then examined by flow cytometry.
Total 20000 cells were counted via FITC channel.The mean fluorescence intensity was analyzed by FlowJo.
Chlorpromazine (an inhibitor of clathrin-mediated endocytosis), nystatin (an inhibitor for caveolin-dependent endocytosis), and wortmannin (a macropinocytosis inhibitor) were selected to investigate the endocytosis mechanisms.Firstly, CT26 cells were seeded on a confocal dish for 12 hours, and then incubated with corresponding inhibitors (chlorpromazine 20 µg mL -1 , nystatin 20 µg mL -1 , Wolman penicillin 1 µg mL -1 ) for 1 h.Then WP5⊃D/DL/L-Arg+DOX+ICG was added and the mixture was incubated at 37 ℃ for 24 hours.After incubation, the cells were washed three times with PBS and the nuclei were stained with DAPI for 20 minutes.The cellular uptake results were then observed by confocal fluorescence microscopy.
Photothermal performance.WP5⊃D/DL/L-Arg+DOX+ICG solutions were exposed to 808 nm laser for 10 minutes, and the temperature changes of WP5⊃D/DL/L-Arg+DOX+ICG solutions were monitored separately by a thermocouple thermometer.
To investigate the photothermal stability of WP5⊃D/DL/L-Arg+DOX+ICG, WP5⊃D/DL/L-Arg+DOX+ICG solution (500 µg mL −1 ) were irradiated with an 808 nm laser for 10 minutes, then the laser was turned off and the solution was cooled at room temperature for 10 minutes.Six laser on/off cycles were performed.
To visually detect the photothermal behavior, upon the 808 nm laser irradiation, the temperature of WP5⊃D/DL/L-Arg+DOX+ICG solution was monitored by a thermal imager (Fluke, Ti-959 Hz).The thermal images were taken every 2 minutes.
Cytotoxicity tests.The cytotoxicity of WP5⊃D/DL/L-Arg, WP5⊃D/DL/L-Arg+DOX, WP5⊃D/DL/L-ICG and WP5⊃D/DL/L-Arg+DOX+ICG were evaluated by the CCK-8 method.Specifically, the CT26 cells were inoculated in a 96-well plate at a density of 1×10 4 cells per well at 37 °C and 5% CO2 for 24 hours.Then, the cells were incubated with different concentrations of WP5⊃D/DL/L-Arg, WP5⊃D/DL/L-Arg+DOX, WP5⊃D/DL/L-ICG and WP5⊃D/DL/L-Arg+DOX+ICG (37 °C, 24 hours), respectively, for 24 hours.In the end, CCK-8 solution was added to each well, and the plates were incubated for another 4 hours.After that, a microplate reader was used to measure the UV-Vis absorbance at 450 nm.
Hoechst/PI Co-staining experiments.The antitumor activity of WP5⊃D/DL/L-Arg+DOX+ICG was evaluated by Hoechst/PI co-staining strategy.Specifically, the cell suspension was diluted and added to a 24 well plate where the cell slides have been placed.The cell density was 20000 cells per well.The above 24 well plate was incubated at 37 ℃ for 24 hours.Then the WP5⊃D/DL/L-Arg+DOX+ICG (50 µg mL −1 ) were added and further incubated at 37 ℃ for 24 hours.For laser groups, after WP5⊃D/DL/L-Arg+DOX+ICG were added, The mixtures were exposed to 808 nm laser for 10 min and further incubated at 37 ℃ for 24 hours.After that, the cells of laser groups and other groups were stained with Hoechst/PI for 20 minutes, followed with the washing with sterile PBS for three times.Eventually, the cells on the slides were observed by confocal fluorescence microscopy.

Evaluation of cellular mitochondria potential.
To monitor the mitochondrial membrane potential of CT26 cells after various treatments, the CT26 cells were inoculated onto a confocal dish for 2 hours.After 24 hours of incubation with WP5⊃D/DL/L Arg+DOX+ICG, CT26 cells were stained with JC-1 at 37 °C for 20 minutes.After carefully washing according to the manufacturer's instructions, the CT26 cells on the slide were observed by confocal fluorescence microscopy.Histology Analysis.After 14 days of various treatments, the main organs (heart, liver, spleen, lungs, and kidneys) and tumors of the mice were removed and placed in PBS containing 4% paraformaldehyde.After that, the samples were embedded in paraffin, cut into 3 µm thickness, stained with hematoxylin and eosin (H&E), and then histopathology analysis was performed with optical microscope.

In vivo
Statistical Analysis.The experimental results were expressed as mean ± SD.The significance of differences was analyzed using two-tailed unpaired student's t-test and one-way analysis of variance (ANOVA) test.Differences between datasets were considered to be significant at p value <0.05.
was dialyzed in ultrapure water to remove the unloaded ICG.The concentration of WP5⊃D/DL/L-Arg+ICG mentioned in this work is referred to the concentration of Arg.Preparation of WP5⊃D/DL/L-Arg+DOX+ICG.To prepare WP5⊃D/DL/L-Arg+DOX+ICG, DOX and ICG (molar ratio of DOX: ICG: WP5⊃D/DL/L-Arg = 1:1:2) were added to 10 mL of WP5⊃D/DL/L-Arg solution, respectively, and stirred for 24 hours.After that, WP5⊃D/DL/L-Arg+DOX+ICG solution was loaded into dialysis bag (molecular weight cutoff 14000), and was dialyzed in ultrapure water to remove the unloaded DOX and ICG.The aqueous solution outside the dialysis bag was changed periodically until the aqueous solution outside the dialysis bag showed negligible UV-Vis absorption of both DOX and ICG.The aqueous solution outside the dialysis bag was collected and measured by UV-Vis spectrometer.Then, the encapsulated amounts of DOX and ICG were calculated according to the standard calibration curve (yDOX = 0.01771x + 0.01448, yICG = 0.11401x + 0.11733).The drug encapsulation efficiency of DOX and ICG loaded in WP5⊃D/DL/L-Arg were calculated according to the following formula: Encapsulation efficiency = mass of loaded drug/total amount of drug input.The concentration of WP5⊃D/DL/L-Arg+ICG mentioned in this work is referred to the concentration of Arg.
chemo-photothermal cancer therapy.Male Balb/c mice were purchased from Yangzhou University Comparative Medicine Centre.The in vivo experiments were authorized by the Science and Technology Department of Jiangsu Province and conducted with the approval of the Medical Ethics Committee of Yangzhou UniversityMedical Academy(YXYLL-2022-44).To evaluate the cancer therapeutic efficacy of WP5⊃D/DL/L-Arg+DOX+ICG, four-week old male Balb/c mice were selected and fed until their body weight was greater than 20 g.Then, 100 µL of the cultured CT26 cell solution was piped and injected subcutaneously into the back of the mice.After one week, when the tumor volume increased to ~250 mm 3 , the CT26 tumor bearing mice were divided into four groups (n = 5 in each group) and treated with the PBS, .In each case, 100 µL (500 µg/mL) of corresponding solution was injected into the tail vein of mice every two days.For groups 2, 3, and 4, after 3 hours' injection, the mice were subjected to 10 minutes' laser irradiation (808 nm, 1.0 W/cm 2 ) on the tumor area, respectively.Under NIR light irradiation, the temperature changes of tumor sites in each group of mice were monitored using an infrared thermal imager (Fluke, Ti-959 Hz).To measure the length and width of the tumor, a digital caliper was used.According to the equation: V (mm 3 ) = length × width 2 /2, the size of the tumor can be calculated.The tumor inhibition rate= (Vcontrol − VD/DL/L)/Vcontrol × 100％, Vcontrol refers to the average tumor volume in the control group on day 14, VD/DL/L refers to the average tumor volume in the WP5D/DL/L-Arg+DOX+ICG group on day 14.

Fig. S4 .
Fig. S4. of H1 of WP5 against the concentration of (a) DL-Arg and (b) L-Arg.The red lines were obtained from non-linear curve-fitting.

Fig. S9 .
Fig. S9.ITC results of the addition of D-Arg into liposome dispersion.